I prepared the LPS solution in PBS at 5 mg/mL. Then I used 0.22um filter in order to remove the contaminate for cell study. I found it was hard to purify the solution for it was hazy. So, should I prepare the LPS in DMEM with working solution (50 ng/mL) and then filtration, or do not filtration? The other question is about the subpackages of stock LPS solution. Should I use the silanized or glass containers?
I have worked with LPS for over 20 years, and there are a few basic things to remember when you prepare LPS for use in experimental protocols. First is that LPS does not really go into solution, but is actually just in suspension. Therefore it is important to vortex your stock immediately before making any dilutions, adding diluted LPS to medium or administering to animals. Second is that it is not stable, and stocks cannot be stored indefinitely or frozen and thawed. We have had the best and most reproducible results by preparing a 1 mg/ml stock in HBSS and storing it in single use aliquots at -20. Lyophilized LPS can be stored refrigerated for years, but once reconstituted, stocks are discarded after 3 months at -20. We buy tissue culture grade LPS from Sigma, reconstitute the entire bottle at once, and prepare it with sterile diluents in sterile tubes, so rarely see a need to filter, especially since most incubations of LPS with cells are for less than 24h. It is generally not a good idea to filter LPS preparations, but if you feel it is absolutely necessary, filter the final media to be added to cells so the LPS is in its most diluted state. Hope this helps!
Hi! I recommend to dissolve LPS directly in cell culture media. When I was stimulating PBMC and SMC with LPS (used RPMI1640 and MEM) I made a stock the same day in eppis. It works well. Good luck!
Regarding the haziness of your solution: Have you checked the manufacturer's / distributor's instructions. Sigma for instance indicates a maximum solubility of 5 mg/mL in H2O but mentions that the solution will be slightly hazy. So maybe it's normal? Try preparing a lower concentration if you're concerned?
Regarding the storage you find good information at the bottom of this document(https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Datasheet/6/62325dat.pdf). I'm not sure how much these things differ between LPS from different strains.
I have used LPS from several companies and have the same, it will be hazy if you dissolve it at maximum solubility that's normal. It will go clear when you further dilute it to use in experiments. I normally make my stock 5mg/ml in PBS and aliquot in to stocks in small eppendorfs for freezing down at -20C, I aliquot into 100ul as I use roughly 90ul of LPS for each experiment so its handy just to have to pull out one stock tube. I then dilute down to 50 & 100ng/ml in media fresh when I am doing experiments. When you dilute down again in media the media will be clear and the haze disappears. You can filter it if you want but I don't since my medias filtered twice by the time I get to experiments anyway!
For subpackages for storing have you already dissolved the LPS in PBS? If you have I would suggest alliquotting into eppendorfs and storing at -20C. Just make sure your eppendorf tubes are sterile, I would advise you autoclave them before use and autoclave the PBS before yit aou use s well if its homemade, if its in sterile bottles form a manufacturer you will be fine using it from the bottle. The main thing thing you really want to do is avoid repeated freeze thaw cycles with your LPS as this can damage the integrity of the molecule. What volumes you aliquot is dependent really on what volumes you are using for an experiment and what is convenient for yourself.
Good luck with your experiments!
I have worked with LPS for over 20 years, and there are a few basic things to remember when you prepare LPS for use in experimental protocols. First is that LPS does not really go into solution, but is actually just in suspension. Therefore it is important to vortex your stock immediately before making any dilutions, adding diluted LPS to medium or administering to animals. Second is that it is not stable, and stocks cannot be stored indefinitely or frozen and thawed. We have had the best and most reproducible results by preparing a 1 mg/ml stock in HBSS and storing it in single use aliquots at -20. Lyophilized LPS can be stored refrigerated for years, but once reconstituted, stocks are discarded after 3 months at -20. We buy tissue culture grade LPS from Sigma, reconstitute the entire bottle at once, and prepare it with sterile diluents in sterile tubes, so rarely see a need to filter, especially since most incubations of LPS with cells are for less than 24h. It is generally not a good idea to filter LPS preparations, but if you feel it is absolutely necessary, filter the final media to be added to cells so the LPS is in its most diluted state. Hope this helps!
i am trying to find a proper silanized containers to store LPS if anybody can direct me .thanks
Big thanks to all contributors. This thread enabled me in reconstituting LPS for a current study.
Is there any fear of bringing the endotoxin LPS into your tissue culture hood and contaminating cultures, I hear it is very sticky....
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